Antimicrobial resistance is the response of microbes to the selective pressure of an antimicrobial drug therapy. Concerns over increased resistance and the emergence of multidrug resistant strains of gram-negative bacteria (Pseudomonas, Klebsiella, Enterobacter, Acinetobacter, Salmonella species) and gram-positive organisms (Staphylococcus, Enterococcus, Streptococcus species) have resulted in an increased number of reports encouraging a reduction in the use of antibacterials, appropriate choice of antibacterials and regimens, prevention of cross-infection and development of new antibacterials (Sharma, et al., Indian J Med. Sci. 9(3):120-9 (2005)). Resistance to some antimicrobial drugs can be overcome by modifying the dosage regimens (e.g., using high-dose therapy) or inhibiting the resistance mechanism (e.g., beta-lactamase inhibitors), whereas other mechanisms of resistance can only be overcome by using an agent from a different class.
Malaria, for example, is the most prevalent and deadliest parasitic disease worldwide (Anonymous, World Malaria Report. (World Health Organization) (2010) (Murray, et al., Lancet, 379(9814):413-431 (2012)). Although once eradicated in many developed countries, malaria is reemerging and spreading in to new areas, such as Central Asia, and Eastern Europe. More people are now dying of malaria than thirty years ago (Malaria Foundation International, malaria.org). Although the traditional drug regime of chloroquine is safe and inexpensive, resistance to the drug is high or on the rise in Asia, and parts of Africa and South America. In some areas of Asia there is resistance to all the major drugs.
Malaria is caused by intraerythrocytic protozoan parasites of the genus Plasmodium. Four species, P. falciparum, P. vivax, P. malariae and P. ovale are known to be infectious to humans, and more recent cases of infection due to P. knowlesi have also been reported (Singh, et al., Lancet 363(9414):1017-1024 (2004)). The lack of a universally effective vaccine to combat this disease and the growing spread of resistance to all currently known antimalarials, including those used in combination therapy, emphasize the need for new approaches to develop new therapies that specifically target essential functions of the parasite to block both infection and transmission.
The genome of P. falciparum has 23 Mb and encodes approximately 5300 proteins, the majority of which are of unknown function and their importance in parasite development remains to be determined. This is due mostly to the difficulty of genetically manipulating this organism using forward genetic approaches, lack of a RNAi machinery and absence of regulatable promoters. Current techniques for genetic disruption are mostly useful for those genes that are not essential for blood stage development (Ullu, et al., Cell Microbiol 6(6):509-519 (2004) El Bissati, et al., Proc Natl Acad Sci USA 103(24):9286-9291 (2006)). Consequently, only a limited number of genes have been shown through genetic means to play an essential role in parasite development and thus are valid targets for development of new antimalarial drugs. Nutritional complementation has recently been used to generate a conditionally lethal P. falciparum mutant lacking the primary purine transporter PfNT1 (El Bissati, et al., Proc Natl Acad Sci USA 103(24):9286-9291 (2006)). However, this approach cannot be used widely to create conditionally lethal mutants. Although new methods for inducible expression have been reported, they have had limited success in creating stably inducible knockouts (Armstrong, et al., Nat Methods 4(12):1007-1009 (2007), Meissner, et al., Proc Natl Acad Sci USA 102(8):2980-2985 (2005), Muralidharan, et al., Proc Natl Acad Sci USA 108(11):4411-4416 (2011), Russo, et al., Proc Natl Acad Sci USA 106(5):1554-1559 (2009), Dvorin, et al., Science 328(5980):910-912 (2010), Farrell, et al., Science 335(6065):218-221 (2012)).
One class of compounds under investigation as a complement or alternative to small molecule antimicrobial drugs is inhibitory nucleic acids. Several inhibitory nucleic acid methods involving RNA, or some chemically modified form of RNA or DNA (siRNA, hammerhead RNA, antisense RNA, LNA or PNA) are being employed in attempts to inhibit the expression of genes, including essential microbial genes, or drug resistance genes in vivo. Among these are basic peptide-morpholino oligonucleotide conjugates. Some practical success has been observed, in particular, in terms of treating mice infected with bacteria and prevention of infection of mammalian tissue by dangerous viruses (Jepsen et al., 14:130-46 (2004), Oh et al., Mol. Cell. 28:341-345 (2009), Lanford et al., Science 327:198-201 (2010), Mellbye et al., Antimicrob Agents Chemother 53:525-530 (2009), Tilley et al., Escherichia coli. J Antimicrob Chemother 59:66-73 (2007), Warren et al., Nature Medicine 16:991-994 (2010), Soler et al., Proc Natl Acad Sci USA 106:13230-13235 (2009), Mellbye et al., J. Antimicrob. Chemoth. 65:98-106 (2010), Greenberg et al., J Infect Dis 201:1822-1830 (2010)). Despite these limited successes, there is a need to improve the safety and efficacy of inhibitory nucleic acid strategies before they can be successfully translated to the clinic.
Accordingly, it is an object of the invention to provide improved inhibitory nucleic acids with greater efficacy that can be used at a lower dosage.
It is also an object of the invention to provide alternative compositions and methods for treating microbial infections, particularly drug resistant infections such as malaria.
It is another object of the present invention to provide compositions for preventing development of drug resistance in bacteria and parasites such as malaria.
It is also an object of the invention to provide compositions and methods for functional analysis of microbial genomes, such as those of Plasmodium. 